Wx Gene Editing via CRISPR/Cas9 System in Rice

doi:10.16819/j.1001-7216.2018.7061

Abstract

Abstract:

【Objective】 Amylose content is closely related to rice quality. The objective of this research is to knockout Wx gene for obtaining rice mutants with suitable amylose content.【Method】 Vector pGK03-Wx-gRNA, a CRISPR/Cas9 system, targeted either the first (target 1) or the second (target 2) exon of Wx gene was constructed and transformed into super-rice Chujing 27 to edit the Wx gene. Independent transformants were obtained via Agrobacterium-mediated transformation with hygromycin selection. The transgenic lines of T₀, T₁ and T₂ generations were identified by DNA sequencing, in addition to qPCR and amylose content assay of T₁. 【Result】 Nine T₀ independent transgenic lines were generated, the L1-L5 from target 1 and L6-L9 from target 2. Eight lines (L1-L8) were mutants and L9 was not confirmed as mutant by sequencing. Therefore, the mutation rate was 100% for target 1, and 75% for target 2. T₁ and T₂generations of the eight lines (L1-L8) were further sequenced, which revealed two types of mutation: base deletion (single base, double and up to fourteen bases), and base insertion. Some lines (L1, L2, L3 and L6) were reedited at T₁ generation when compared to its T₀ generation, while the rest lines were stable at the mutation sites. All the eight mutants were stable when comparing their target gene sequence at T₁ and T₂ generations. qPCR results indicated that Wx gene expression at RNA level was considerably reduced in the mutant. The seed amylose content was reduced from 17.5% to 1.93%(P<0.01). 【Conclusion】 The Wx gene was successfully edited by CRISPR/Cas9 system in rice, the inheritable mutants with average amylose content at 1.93% were obtained, which would provide valuable germplasm for rice quality improvement.

Key words: rice, CRISPR/Cas9 system, Wx gene, gene editing, amylose content

摘要：

【目的】直链淀粉含量与稻米品质密切相关。Wx基因是控制水稻直链淀粉合成的主效基因,通过对Wx基因定点编辑以获得稳定遗传、直链淀粉含量适宜的突变体。【方法】构建CRISPR/Cas9表达载体pGK03-Wx-gRNA (靶点1和2分别在Wx基因第1和第2外显子),利用工程菌EHA105遗传转化超级稻楚粳27,潮霉素筛选获得转化株系,对转化株系及其后代进行分子检测、测序、基因表达和遗传稳定性分析以及直链淀粉含量测定。【结果】获得9个独立的T₀代转化株系,靶点1(L1~L5) 5个株系,突变频率100%,靶点2(L6~L9) 4个株系,突变频率75%。由T₀代突变体衍生出T₁和T₂代株系,测序发现T₀、T₁和T₂代株系出现缺失(单、双、多碱基缺失)和单碱基插入两种突变类型;T₀至T₁代部分株系(L1、L2、L3和L6)发生再编辑,T₁至T₂代遗传稳定。与野生型相比,突变株系RNA水平Wx基因表达量显著下降(P<0.01),稻米直链淀粉含量显著降低(P<0.01),从17.5%降到1.93%。【结论】利用CRISPR/Cas9系统成功编辑水稻Wx基因,获得了稳定遗传、低直链淀粉含量的突变体,为稻米品质改良提供了材料。

关键词: 水稻, CRISPR/Cas9系统, Wx, 基因编辑, 直链淀粉含量

CLC Number:

Bingkun WANG, Hui ZHANG, Ruke HONG, Jinwen ZHANG, Rui YANG, Qiong LUO, Qianchun ZENG. Wx Gene Editing via CRISPR/Cas9 System in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(1): 35-42.

汪秉琨, 张慧, 洪汝科, 张锦文, 杨睿, 罗琼, 曾千春. CRISPR/Cas9系统编辑水稻Wx基因[J]. 中国水稻科学, 2018, 32(1): 35-42.

Figures/Tables 10

Table 1 Oligo sequence and test primer sequence.

Oligo名称	序列	检测引物	序列
Oligo name	Sequence	Test primer	Sequence
Oligo-Target1-F	TGTGTGGGCGAATGGCCACAGGGTCA	G-Target1-F	GCATGAACGTCGTGTTCGTC
Oligo-Target1-R	AAACTGACCCTGTGGCCATTCGCCCA	G-Target1-R	CCTCACCCTCTCGTACCTGT
Oligo-Target2-F	TGTGTGGCTTGAGGCCCTGGAACCCG	G-Target2-F	TGCAGAGATCTTCCACAGCA
Oligo-Target2-R	AAACCGGGTTCCAGGGCCTCAAGCCA	G-Target2-R	TTGAAGACGACGACGGTCAG

Fig. 1. Positions of the two targets in Wx gene.

Fig. 2. Vector map of pGK-Wx-gRNA. LB, Left border; U6,Rice U6 promoter; SG, Guide RNA; UBI, UBI promoter; NOS Ter, NOS terminator; 35S, 35S promoter; Hpt, Hygromycin gene; PolyA Ter, PolyA terminator; RB, Right border.

Fig. 3. PCR results of transformants L1-L9.

Fig. 4. DNA sequence alignment for wild type and the nine transformants in T0 generation. sequences are shown by black hyphens. One base deletion is recorded by minus one and the one base insertion by plus one. The targeted sequence is highlighted with underline and the PAM sequence in shadow. Mutation with 1 bp insertion is represented by a, the deleted

Fig. 5. Amino acid sequence alignment for wild type and nine transformants in T0 generation. The deleted amino acids are shown by black hyphens, the translation were terminated earlier in mutants L1-L5 and L6-L8.

Fig. 6. DNA sequence alignment for the wild type and the L1-L8 in T1 and T2 generations. The targeted sequence is highlighted with underline and the PAM sequence in shadow. Mutation with 1 bp insertion is represented by a, the deleted sequences are shown by black hyphens.

Fig. 7. Relative expression level of Wx gene in wild type and transformants in T1 generation(Mean±SD). **indicates significant difference at P<0.01(t test).

Table 2 Amylose content comparison of the wild type and transformants L1-L8.

株系 Line	WT	L1	L2	L3	L4	L5	L6	L7	L8	平均 Average
直链淀粉含量Amylose content/%	17.5	1.78^**	1.82^**	1.76^**	2.15^**	2.06^**	2.04^**	1.93^**	1.87^**	1.93

Fig. 8. Brown rice of wild type and mutants. WT, Brown rice of Chujing 27; L1-L8, Brown rice of mutants in T1 generation and the bar represents one centimeter.

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